Open Access

BOTRYTIS-INDUCED KINASE1, a plasma membrane-localized receptor-like protein kinase, is a negative regulator of phosphate homeostasis in Arabidopsis thaliana

BMC Plant BiologyBMC series – open, inclusive and trusted201616:152

https://doi.org/10.1186/s12870-016-0841-1

Received: 27 January 2016

Accepted: 28 June 2016

Published: 7 July 2016

Abstract

Background

Plants have evolved complex coordinated regulatory networks to cope with deficiency of phosphate (Pi) in their growth environment; however, the detailed molecular mechanisms that regulate Pi sensing and signaling pathways are not fully understood yet. We report here that the involvement of Arabidopsis BIK1, a plasma membrane-localized receptor-like protein kinase that plays critical role in immunity, in Pi starvation response.

Results

qRT-PCR analysis revealed that expression of BIK1 was induced by Pi starvation and GUS staining indicated that the BIK1 promoter activity was detected in root, stem and leaf tissues of plants grown in Pi starvation condition, demonstrating that BIK1 is responsive to Pi starvation stress. The bik1 plants accumulated higher Pi content in root and leaf tissues and exhibited altered root architecture such as shorter primary roots, longer and more root hairs and lateral roots, as compared with those in the wild type plants, when grown under Pi sufficient and deficient conditions. Increased anthocyanin content and acid phosphatase activity, reduced accumulation of reactive oxygen species and downregulated expression of Pi starvation-induced genes including PHR1, WRKY75, AT4, PHT1;2 and PHT1;4 were observed in bik1 plants grown under Pi deficient condition. Furthermore, the expression of PHO2 was downregulated while the expression of miRNA399a and miRNA399d, which target to PHO2, was upregulated in bik1 plants, compared to the wild type plants, when grown under Pi deficient condition.

Conclusion

Our results demonstrate that BIK1 is a Pi starvation-responsive gene that functions as a negative regulator of Pi homeostasis in Arabidopsis.

Keywords

BOTRYTIS-INDUCED KINASE1 (BIK1) Phosphate starvation stress Phosphate starvation response Root architecture

Background

Phosphate (Pi) is one of the indispensable macronutrients to plants for growth, development and reproduction. Pi deficiency (−Pi) is one of the main limiting factors for increasing crop yield and improving quality because of low bioavailability of Pi in soil [14]. To cope with Pi deficiency, plants develop a series of tightly controlled adaptive responses including external developmental alterations of increasing Pi absorption and internal metabolic, physiological and biochemical alterations of reducing Pi usage [46]. To increase Pi uptake under Pi depletion stress, plants adapt to modulate the root architecture bearing more and longer lateral roots as well as denser root hairs, which enable the roots to explore the Pi resources in soil [712]. To reduce Pi consumption under Pi deficiency condition, plants often modulate metabolisms to maintain intracellular Pi homeostasis by reducing metabolic consumption of Pi [1, 3, 1315] and degrading compounds to release Pi [16]. Furthermore, a series of physiological and biochemical adaptions including the induction and secretion of phosphatases and organic acids and accumulation of protective metabolites such as anthocyanin help augment the availability of both endogenous and exogenous Pi [1, 4, 17, 18].

Genetic, physiological and biochemical studies in Arabidopsis have demonstrated that the acquisition, allocation, and metabolism of Pi are highly regulated processes and require the concerted action of multiple membrane Pi transport systems [1923]. Among five distinct classes of proteins possessing Pi transport activity, the plastidic Pi translocator group function as antiport systems, whereas the other four Pi transporter families, named the PHOSPHATE TRANSPORTER1 (Pht1), Pht2, Pht3 and Pht4, contribute to the acquisition, allocation and remobilization of Pi [2427]. Most of the plasma membrane-localized high affinity transporters in the Pht1 family mediate Pi acquisition from external environment [1, 24, 2830]. Once Pi is transported into root epidemic cells, translocation and allocation of Pi within the plants and cells are key steps in maintaining Pi homeostasis at cell and whole plant levels. The low affinity transporters in the Pht2 and Pht4 families are thought to participate mainly in Pi transfer across internal cellular membranes and thus allocate Pi in different compartments of the cells [25, 27, 3133]. Pht1;5, a Pht1 family member, PHO1, Pht1;8 and Pht1;9 were shown to play critical roles in systemic regulation of Pi homeostasis, e.g. mobilization of Pi from source to sink organs in accordance with the Pi status of the plant [3436].

Functional characterization of genes in a number of mutants with altered response to Pi depletion have led to the identification of several different regulatory mechanisms controlling the adaptive responses to Pi starvation. These regulatory mechanisms include transcriptional regulation by transcription factors such as PHR1, WRKY6, WRKY42, WRKY45, WRKY75, ZAT6, bHLH32, ERF070 and MYB62 [3747], posttranscriptional regulation by microRNAs including miRNA399 [4853], posttranslational regulation by protein modifications such as sumoylation of SIZ1 [54], phosphorylation of Pht1.1 and activation of MKK9-MPK3/MPK6 module [55, 56], deubiquitination of UBP14 [57] and chromatin histone modification and epigenetic [46, 58]. Furthermore, it was also demonstrated that Pi starvation response cross-talks signaling mediated by different hormones such as auxin [10, 15, 5961], cytokinin [10, 15, 62, 63], ethylene [9, 10, 6466] and gibberellin [67]. Although great advance on the adaptive response to Pi starvation has been made during the last decade, the molecular mechanism that regulates these adaptive processes has yet to be elucidated in detail.

The Arabidopsis thaliana BOTRYTIS-INDUCED KINASE1 (BIK1) encodes a plasma membrane-localized receptor-like protein kinase and plays critical roles in Botrytis cinerea resistance [68]. Recent studies have shown that BIK1 interacts with receptors for pathogen- or damage-associated molecular patterns such as FLS2 and PEPRs to regulate immune response against different types of pathogens [6973] and defense response to insect pests [74]. The bik1 mutant plants showed an altered root architecture [68], similar to morphological phenotypes often seen in mutants with Pi starvation response [75], indicating a possible involvement of BIK1 in Pi starvation response in Arabidopsis. Therefore, we investigated whether BIK1 functions in Pi starvation response and our results demonstrate that BIK1 plays a role in regulation of Pi homeostasis in Arabidopsis.

Results

Responsiveness of BIK1 to Pi starvation

When grown on MS medium under normal conditions, the bik1 plants produced shorter primary roots and longer and significantly more root hairs and lateral roots than WT plants [68], which is reminiscent of the mutants with defects in Pi nutrition [75]. These observations led us to examine whether BIK1 has a function in Pi starvation response. We first examined whether BIK1 is responsive to Pi starvation stress by analyzing the expression patterns of BIK1 in seedlings grown under + Pi and –Pi conditions. As shown in Fig. 1a, expression of BIK1 was detected in roots, shoots and leaves of seedlings grown under + Pi condition and no significant changes in BIK1 expression was observed during the experiment period. However, expression level of BIK1 was markedly induced with similar patterns in roots, shoots and leaves of seedlings after transferring to medium without Pi supplement (Fig. 1a). The transcript levels of BIK1 in seedlings grown under –Pi condition increased at 12 h and peaked at 24 and 48 h after transferring, leading to 7.5 ~ 11.2 folds of increases over those in seedlings grown under + Pi condition (Fig. 1a). To gain further information on spatial expression of the BIK1 gene in response to Pi starvation, we generated BIK1 pro ::GUS transgenic lines and compared the GUS staining patterns in T2 seedlings during Pi starvation stress. Slight GUS staining was observed in leaves and roots of the BIK1 pro ::GUS seedlings at 0 h after transferred to medium without Pi supplement (Fig. 1b), indicating a basal expression of BIK1 in leaf and root tissues, similar to the results from RT-PCR. At 1 day after transferred to medium without Pi supplement, significant GUS staining was easily seen in roots, shoot and leaves of the BIK1 pro ::GUS seedlings (Fig. 1b). qRT-PCR analysis showed 7.3 and 9.7 folds of increases in the expression levels of GUS gene in root and leaf tissues of the BIK1 pro ::GUS seedlings at 1 day after transferring to –Pi condition (Fig. 1c). Notably, GUS staining was clearly observed in vascular tissues of roots, shoots and leaves of the BIK1 pro ::GUS seedlings subjected to Pi starvation (Fig. 1b). Together, data from RT-PCR and GUS staining demonstrate that BIK1 is responsive to Pi starvation.
Fig. 1

Responsiveness of BIK1 to Pi starvation. a Expression changes of BIK1 in different tissues of WT plants under + Pi and –Pi conditions. Seven-day-old seedlings grown hydroponically under normal Pi condition were transferred to medium supplemented with Pi (+Pi, 250 μM) or without Pi (−Pi). Roots, shoots and leaves were collected for analysis of BIK1 expression by qRT-PCR at indicated time points after transferring. b Detection of BIK1 promoter activity in BIK1 pro ::GUS seedlings by GUS staining. c Expression changes of GUS gene in root and lead tissues of BIK1 pro ::GUS seedlings under –Pi condition. Seven-day-old BIK1 pro ::GUS seedlings grown in normal medium were transferred to medium without Pi supplement and samples were collected at 0 and 1 day after transferring for GUS staining and analysis of gene expression. Data were normalized with the transcript level of UBQ10 and relative expression levels were shown as folds of the UBQ10 level. Data presented are the means ± SD from three independent experiments and * above the error bars indicate significant differences at p < 0.05 level between the + Pi and –Pi conditions

Increased Pi concentration in bik1 plants

We next examined whether loss of BIK1 function affects Pi homeostasis in bik1 plants. Total Pi contents in leaves and roots of WT and bik1 plants grown hydroponically under + Pi (250 μM) and –Pi conditions for 27 days were measured. As shown in Fig. 2, a significant increase in total Pi contents was observed in leaves and roots of bik1 plants as compared to WT plants under both + Pi and –Pi conditions. When grown under + Pi condition, total Pi contents in roots and leaves of bik1 plants were 0.43 and 1.12 times higher over those in WT plants, respectively (Fig. 2a and b). Similarly, when grown under –Pi condition, total Pi contents in roots and leaves of bik1 plants were 0.55 and 1.05 times higher than those of WT plants, respectively (Fig. 2a and b). These data indicate that BIK1 has a function either in Pi uptake or in the transfer of Pi from the roots to the leaves.
Fig. 2

Increased total Pi content in bik1 plants. Total Pi contents in roots (a) and leaves (b) of WT and bik1 plants grown under + Pi (250 μM) and –Pi condition for 27 days were estimated. Data presented are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level between same tissues of WT and bik1 plants grown under same condition

Altered root architecture in bik1 plants

Root architecture plays important roles in maintaining Pi homeostasisn in plants [75]. We compared the root architecture of WT and bik1 seedlings grown under Pi normal (+Pi) and starvation (−Pi) conditions. When compared with WT seedlings, the bik1 seedlings grown under + Pi condition showed shorter primary root and more lateral roots (Fig. 3a, c and d), and this trend was much evident in the bik1 seedlings grown under –Pi condition (Fig. 3a, c and d). When grown under + Pi and –Pi conditions, the elongation rate of the primary and lateral roots of the bik1 seedlings was markedly reduced as compared with WT seedlings (Fig. 3e and f). Furthermore, the bik1 seedlings grown under + Pi and –Pi conditions showed more and longer root hairs than WT seedlings (Fig. 3b, g and h). These data indicate that BIK1 plays an important role in regulating development of root architecture.
Fig. 3

Altered root architecture in bik1 seedlings grown under + Pi and –Pi conditions. Seven-day-old seedlings grown under normal condition were transferred into fresh medium supplemented with (+Pi, 250 μM) or without Pi (−Pi) and grown for another 7 days. a Root architecture in WT and bik1 seedlings. b Root hairs in WT and bik1 seedlings. Fragments of 5 mm from root tips were presented. c to h Comparative parameters for length of primary roots (c), numbers of lateral roots (d), elongation of primary roots (e) and lateral roots (f), and numbers (g) and length (h) of root hairs of WT and bik1 seedlings grown under + Pi and –Pi condition. Data presented are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level

Increased anthocyanin accumulation and acid phosphatase activity in bik1 plants

Accumulation of anthocyanin and increased secretion of acid phosphatases are characteristic symptoms in plants under Pi starvation conditions [3]. We thus examined and compared the accumulation of anthocyanin and activity of acid phosphatase between WT and bik1 plants grown under + Pi and –Pi conditions. When grown on medium under –Pi condition for 5 days, the leaves of the bik1 seedlings turned to be purple while WT seedlings still kept green (Fig. 4a). The anthocyanin contents in bik1 seedlings grown under + Pi and –Pi conditions were much higher than those in WT seedlings, showing 32 % and 105 % increase, respectively (Fig. 4b). No visible staining and significant changes in acid phosphatase activity were detected in WT and bik1 seedlings grown + Pi condition (Fig. 4c and d). When grown under –Pi condition, roots of the bik1 seedlings are stained deep blue while the staining of roots of WT seedlings is much lighter (Fig. 4c). Similarly, activity of acid phosphatase in roots of bik1 seedlings is much higher than that in WT seedlings, grown under –Pi condition, showing an increase of ~65 % (Fig. 4d). These results indicate that the bik1 seedlings secrete higher levels of acid phosphatase than WT seedlings under Pi starvation stress. Collectively, these results suggest that loss of BIK1 function results in typical Pi starvation responses as revealed by the increased anthocyanin contents and acid phosphatase activity.
Fig. 4

Increased levels of anthocyanin accumulation and acid phosphatases activity in bik1 plants. a and b Measurements of anthocyanin contents. Seven-day-old WT and bik1 seedlings grown on 1/2 MS were transferred into MS medium supplemented with Pi (+Pi, 1 mM) or without Pi (−Pi). Photos were taken and anthocyanin contents were measured at 5 days after treatment. c and d Detection of acid phosphatase activity. Ten-day-old seedlings grown in liquid medium were transferred to fresh medium supplemented with Pi (+Pi, 1 mM) or without Pi (−Pi) and covered with a layer of 0.008 % BCIP-containing agarose. Photos were taken and the activity of acid phosphatase in roots was estimated at 5 days after treatment. Data presented are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level between WT and bik1 plants grwon under same condition

Reduced ROS accumulation in bik1 plants under Pi starvation condition

ROS, generated and accumulated in plant response to different types of stresses, has been implicated in many biological processes including biotic and abiotic responses [76, 77]. Therefore, we examined whether mutation in BIK1 gene affects the balance of ROS and hence leads to accumulation of ROS in plants during Pi starvation stress. In NBT staining of superoxide anion, no significant difference was detected between WT and bik1 plants grown under + Pi condition; however, significant accumulation of superoxide anion in leaves, stem and roots of WT and bik1 plants grown under –Pi condition was observed (Fig. 5a). Notably, accumulation of superoxide anion in WT plants was much higher than that in bik1 plants grown under –Pi condition (Fig. 5a). Quantification of H2O2 contents revealed a significant lower level of H2O2 accumulated in bik1 plants than that in WT plants when grown under + Pi condition (Fig. 5b). Accumulation of H2O2 in WT and bik1 plants grown under –Pi condition was markedly increased, leading to 95 and 78 % of increases as compared with those in plants grown under + Pi condition, respectively (Fig. 5b). Under –Pi condition, the content of H2O2 in bik1 plants was approximately 54 % of that in WT plants (Fig. 5b). These results indicate that loss of BIK1 function led to reduced accumulation of ROS in bik1 plants under –Pi starvation stress.
Fig. 5

Reduced accumulation of ROS in bik1 plants. Four-week-old seedlings grown under + Pi (250 μM) and –Pi conditions were collected for detection of superoxide anion and measurement of H2O2. a NBT staining of superoxide anion in WT and bik1 plants grown under + Pi and –Pi conditions. b Quantification of H2O2 contents in WT and bik1 plants grown under + Pi and –Pi conditions. Data presented are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level between WT and bik1 plants grwon under same condition

Altered expression of Pi starvation-induced genes and miRNA399 in bik1 plants

Expression of some well-characterized Pi starvation-induced genes was analyzed and compared between WT and bik1 seedlings that had been transferred into medium with Pi (+Pi) or without Pi (−Pi) for 5 days. When grown under + Pi condition, the expression levels of AT4 [78], WRKY75 [39], PHT1;2 and PHT1;4 [24, 28] in bik1 seedlings were comparable to those in WT seedlings, while the expression of PHR1 [37] in bik1 seedlings was reduced (Fig. 6a). The expression levels of the Pi starvation-induced genes tested were markedly increased in WT and bik1 plants grown under –Pi condition (Fig. 6a). However, significant reductions in the expression levels of these Pi starvation-induced genes was observed in bik1 plants, as compared with those in WT plants, under –Pi condition (Fig. 6a). These results indicate that mutation in BIK1 leads to reduced expression of the Pi starvation-induced genes in plants grown under –Pi condition.
Fig. 6

Down-regulated expression of Pi starvation-induced genes in bik1 plants. a Expression patterns of Pi starvation-induced genes in WT and bik1 seedlings grown under + Pi and –Pi conditions. Seven-day-old seedlings grown under normal Pi condition were treated with Pi (+Pi, 250 μM) or without Pi (−Pi) for 5 days and total RNA were extracted from root tissues at 5 days after treatment. b Expression patterns of PHO2 and miRNA399 in WT and bik1 seedlings grown under + Pi and –Pi conditions. Seven-day-old seedlings grown under normal Pi condition were transferred to medium supplemented with Pi (+Pi, 250 μM) or without Pi (−Pi) and samples were collected at 48 h after treatment. Expression of Pi starvation-responsive genes and miR399s was analyzed by qRT-PCR using specific primers and data were normalized with the transcript level of UBQ10 as an internal control. Data presented are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level

It was found that expression of PHO2, encoding a ubiquitin-conjugating E2 enzyme, was suppressed by miRNA399, which controls Pi homeostasis in plants and whose expression is up-regulated by Pi starvation [4852]. To examine whether PHO2/miRNA399 links to increased accumulation of Pi in bik1 plants, we analyzed the changes in levels of PHO2 expression and two miRNA399 primary transcripts, miRNA399a and miRNA399d, in bik1 seedlings grown under + Pi and –Pi conditions. The expression level of PHO2 and the transcript levels of miRNA399a and miRNA399d were comparable in WT and bik1 seedlings grown under + Pi condition. However, the expression levels of PHO in WT and bik1 seedlings under –Pi condition were significantly reduced whereas the transcript levels of miRNA399a and miRNA399d in WT and bik1 seedlings grown under –Pi condition were markedly increased, as compared with the corresponding seedlings under + Pi condition (Fig. 6b). A further reduction in the expression of PHO2 and a further increase in the transcripts of miRNA399a and miRNA399d in bik1 seedlings was observed as compared with those in WT seedlings grown under –Pi condition (Fig. 6b). Together, these data indicate that the PHO2/miRNA399 plays a role in regulating Pi accumulation in bik1 plants under –Pi condition.

Discussion

The maintenance of cellular Pi homeostasis in plants involves complicated regulatory mechanisms. Several studies have demonstrated that posttranslational modifications such as phosphorylation, sumoylation and ubiquitination of regulatory proteins play critical roles in Pi starvation responses [54, 55, 57]. Recently, genome-wide co-expression analysis identifies dozens of protein kinases as important regulators of Pi deficiency-induced root hair remodeling [79, 80]. It was also found that in vivo phosphorylation activation of PPC1 (phosphoenolpyruvate carboxylase 1) is involved in the metabolic adaptations of Pi starvation [81] and activation of MKK9-MPK3/MPK6 enhances phosphate acquisition in Arabidopsis [56]. These findings indicate an important role for protein kinases in regulating Pi starvation response. In this study, we found that BIK1, a plasma membrane-localized receptor-like protein kinase [68], plays an important role in modulating Pi starvation responses and functions as a negative regulator of Pi homeostasis in Arabidopsis plants. Thus, evidence presented in the current study renders BIK1 for a novel function in Pi starvation response, in addition to its previously reported functions in immunity [6873].

Several lines of evidence presented in this study support that BIK1 functions in Pi starvation response. Earlier study has shown that expression of BIK1 could be induced by infection with B. cinerea as well as treatments with some well-known defense signaling molecules [68]. Our qRT-PCR analysis of BIK1 expression and determination of BIK1 promoter activity in stable BIK1 pro ::GUS transgenic seedlings demonstrate that BIK1 can be induced by Pi starvation and Pi starvation-induced expression of BIK1 in root and shoot tissues initiated earlier than in leaf tissues (Fig. 1a). The pattern of BIK1 expression induced by Pi starvation is similar to most of the Pi starvation-induced genes identified so far, like WKRY75, ERF070 and ZAT6 [39, 40, 44]. Importantly, promoter activity analysis revealed that GUS staining driven by the BIK1 promoter was initiated in vascular tissues of root (Fig. 1b) that is the primary organ that responds to Pi starvation, further confirming the responsiveness of BIK1 to Pi starvation. Thus, it is likely that BIK1 can respond rapidly to the altered Pi status of plants under Pi starvation condition. It was particularly noteworthy that BIK1 is a plasma membrane-localized receptor-like protein kinase and has been shown to be phosphorylated by other kinases (e.g. BAK1 and FLS2) for its full activity in plant immune responses [69, 70]. In this context, it is thus possible that BIK1 is phosphorylated by unknown kinase during Pi starvation stress and phosphorylated BIK1 acts in Pi starvation response, in addition to its transcriptional regulation upon Pi starvation.

The primary root, lateral root, and root hairs are three main components of the root architecture that is critical to absorption of Pi from soil. Low P availability can drastically alter the root architecture by switching the indeterminate growth to determinate growth to promote lateral root growth [75]. Generally, not only the primary roots but also almost all mature lateral roots enter into the determinate developmental program under low Pi condition [82]. The bik1 seedlings showed a significant change in root architecture and the primary root length of bik1 plants was significantly decreased (Fig. 6). This is similar to a common phenomenon observed that the primary root length is decreased significantly due to the Pi starvation-induced determinate growth in primary root [82]. In addition to the change of the primary root, the bik1 plants showed increased number and length of lateral roots and root hairs (Fig. 6), which increase root surface contacting an increased soil volume to explore Pi availability in soil [75]. Similar root architectures were observed in the bik1 plants under + Pi and –Pi conditions (Fig. 6). Thus, it is likely that the effect of BIK1 on the development of root architecture is independent of the Pi status in plants and Pi availability in soil. Nevertheless, the characteristic root architecture observed in bik1 plants suggests that BIK1 is a negative regulator of lateral root and root hair development. This is in agreement with previous observations that the bik1 plants showed some defects in growth and development, e.g. weak stem strength, early flowering and less seed setting [68]. Together, these data further demonstrate that BIK1 is required for normal plant growth and development [68]. It will be interesting to investigate whether the growth and developmental defects in bik1 plants is due to an activated Pi starvation response and if this is the case, the results obtained will further support a cross-talk between Pi starvation response and plant growth/development. It was previously found that overexpression of ZAT6 retards growth and results in typical Pi starvation responses [40].

Accompanied with high contents of total Pi in roots and leaves of bik1 plants under + Pi and –Pi conditions (Fig. 2) are the significant accumulation of anthocyanin (Fig. 4a and b), increased activity of acid phosphatase (Fig. 4a and b) and alterations in expression of Pi starvation-induced genes (Fig. 6). Collectively, these data imply that, whatever grown under + Pi condition or under –Pi condition, a Pi starvation response including those of physiological, molecular and metabolic changes is activated in bik1 plants. Increased activity of acid phosphatases in bik1 plants grown under –Pi condition (Fig. 3b) not only represents a characteristic Pi starvation response [83] but also can release more Pi available for absorption by the roots. This is supported by the findings that mutations in genes encoding for purple acid phosphatases affect markedly the uptake of Pi from exogenous sources [8486]. Because expression of BIK1 was rapidly induced by Pi starvation (Fig. 1), it seems possible that BIK1 is involved in Pi uptake by regulating the activity of acid phosphatase. Similar observations were also obtained for some Pi starvation response regulators such as PHR1 and ZAT6, which have been shown to affect activity of acid phosphatase and Pi uptake in transgenic plants with altered expression of these genes [40, 85, 87, 88]. Interestingly, expression of Pht1;2 and Pht1;4, encoding for high-affinity Pi transporters [24, 28], WRKY75, encoding a WRKY transcriptional factor involved in Pi acquisition [39], At4, a member of the Mt4/TPSI1 gene family involved in Pi distribution [78], and PHR1, encoding a MYB transcriptional factor involved in Pi starvation response signaling [37], was suppressed significantly in roots of bik1 plants under –Pi condition (Fig. 6a). These data demonstrate that BIK1 has global effect on a set of Pi starvation-induced genes that are involved in Pi acquisition and mobilization. Notably, the expression levels of the tested Pi starvation-responsive gene except PHR1 in bik1 plants were slightly reduced as compared to those in WT plants under + Pi condition (Fig. 6a), indicating a possibility that these Pi starvation-responsive genes may be also affected by mutation of BIK1 itself. Particularly, the expression of PHR1 in bik1 plants grown under + Pi condition was significantly reduced as compared to that in WT plants (Fig. 6a), leading to an open question whether BIK1 acts upstream of PHR1 in regulating the Pi starvation signaling. This can be clarified by phenotyping and analysis of PHR1 expression in BIK1-overexpressing plants and/or detailed examination of biochemical and genetic requirement of BIK1 for Pi starvation response. It is currently difficult to link the function of BIK1 to Pi uptake or its root-to-leaf translocation as Pi uptake and transportation in WT plants is also affected under –Pi condition (Fig. 2). This can further be explained by the reduced expression of these tested Pi starvation-induced genes in root of bik1 plants as compared to those in WT plants under –Pi condition (Fig. 6). Furthermore, altered expression patterns of PHO2 and miRNA399a/d, which are thought to be involved in systemic signaling of Pi starvation response and Pi distribution in plants [49, 52], were also observed in bik1 plants grown under + Pi and –Pi conditions (Fig. 6b). Thus, it is likely that BIK1 is involved in maintaining Pi homeostasis in whole plants. Taken together, these data support an idea that BIK1 is a negative regulator of Pi starvation responses, probably through affecting development of root architecture and a series of physiological and biochemical events related to Pi acquisition, mobilization and translocation. It is reasonable to speculate that, under Pi starvation stress, the bik1 plants may experience a reduced Pi content as the WT plants but they can uptake efficiently Pi from growth environment with their significantly increased root surface area, leading to an increased Pi content in root and leaf tissues of bik1 plants (Fig. 2). However, the detailed mechanism that BIK1 functions in Pi starvation response remains to be explored further. Particularly, characterization of the targets that are phosphorylated by BIK1 will be greatly helpful in elucidating the early signaling events that determine Pi starvation response.

ROS not only are toxic compounds produced in plant response to various stresses but also plays an integral role as signaling molecules in regulation of numerous biological processes such as growth, development, and responses to biotic and/or abiotic stress [8991]. The involvement of ROS in Pi starvation response has been established recently. In this study, we found that both WT and bik1 plants grown under –Pi condition accumulated more ROS, represented by H2O2 and superoxide anion, than those in plants grown under + Pi condition (Fig. 5). This is similar to the previous observations that H2O2 concentrations in roots increased upon Pi deprivation [92]. Notably, the accumulation of superoxide anion in WT and bik1 plants under + Pi condition was comparable, the accumulation of superoxide anion in bik1 plants was significantly reduced as compared to that in WT plants under –Pi condition (Fig. 5). By contrast, the bik1 plants accumulated lower level of H2O2 than WT plants grown under + Pi condition (Fig. 5), which is similar to the histochemical staining in soil-grown bik1 plants [68]. Thus, it is likely that BIK1 affects the accumulation of superoxide anion and H2O2 in different ways: loss of BIK1 function suppressed, at least partially, the Pi starvation-induced accumulation of superoxide anion while BIK1 regulates directly the accumulation of H2O2 in plants under normal condition. This may also imply different functions of superoxide anion and H2O2 in Pi starvation response. On the other hand, increased accumulation of ROS in plants under –Pi condition might be one of the stress responses as those in response to other abiotic stress [90]. It was recently shown that localization pattern of ROS accumulated in root during Pi starvation stress is critical to shape the root architecture [9396]. The reduced accumulation of ROS in bik1 plants in relative to those in WT plants (Fig. 5), which is similar to the observation that loss of BIK1 function impaired the PAMP-induced ROS burst in immunity [97, 98], may attribute to the altered root architecture of bik1 plants (Fig. 3). However, detailed analysis of ROS localization and the possible mechanism regulating ROS generation in root of bik1 plants will provide new insights into the connection between BIK1 and ROS in development of root architecture.

Conclusions

In summary, this study demonstrates that BIK1 is a Pi starvation-responsive gene and functions as a negative regulator of Pi homeostasis in Arabidopsis. This not only renders a novel function for BIK1 but also strengthens our understanding of post-transcriptional regulation during Pi starvation responses in plants. Considering that BIK1 is a plasma membrane-localized receptor-like protein kinase, it thus may play a role in sensing and/or processing of the Pi starvation signal during early stage of Pi starvation stress.

Methods

Plant materials and growth condition

Arabidopsis thaliana ecotype Columbia (Col-0) and bik1 (provided by Dr. Tesfaye Mengiste, Purdue University, USA) mutant [68] were used in this study. Seeds were surface sterilized with 70 % ethanol, washed with sterilized distill water and vernalized at 4 °C for 2 days before germination. Hydroponic, solid medium and liquid medium cultivation were used for different purpose of experiments. For solid medium cultivation, the basic medium used contained 2.06 mM NH4NO3, 1.88 mM KNO3, 0.31 mM MgSO4, 0.1 mM MnSO4, 0.03 mM ZnSO4, 0.1 mM CuSO4, 0.3 mM CaCl2, 5.0 mM KI, 0.1 mM CoCl2, 0.1 mM FeSO4; 0.1 mM EDTA, 0.1 mM H3BO3, 1 mM Na2MoO4.2H2O, 3 g/L sucrose, 10 g/L agar, pH 5.8. When treated for Pi sufficiency (+Pi) and Pi deficiency (−Pi), the medium was supplemented with 1 mM KH2PO4, 10 μM KH2PO4 and 0.99 mM KCl. For hydroponic cultivation, seedlings at 5 ~ 7-leaf stage were transferred to 1/2 Hoagland solution for short adaption and then transferred to hydroponic solution containing 250 μM Pi or no Pi [99]. For liquid medium cultivation, seeds were dispensed in 1/2 MS medium without agar and rinsed with sterilized distilled water at 7 days. Seedlings were then transferred in MS liquid medium with Pi (1 mM) or without Pi and allowed to grow with shaking at 85 rpm. Plants were grown in a growth room under a 16 h light (100 μmol · s−1 · m−2 photons m−2 sec−1 of intensity) and 8 h dark cycle at 22 ± 2 °C with 60 % relative humidity.

Measurement of root system architecture

Seven-day-old seedlings grown on 1/2 MS medium were transferred to Petri dishes and treated for + Pi or –Pi for another 7 days. Length of main root, number and length of the lateral root were measured. Total numbers of root hairs in a 5 mm region from root tip were recorded. Data were recorded from 15 individual plants from each treatment.

Anthocyanin analysis

Measurement of anthocyanin was performed as described previously [100]. Briefly, seedlings grown under + Pi or –Pi condition in solid medium were put into 20 mL extraction solution (propanol : HCl : H2O = 18:1:81, V/V/V) and boiled for 90 s. The mixtures were kept overnight in dark at room temperature, centrifuged for 40 min at 5000× g, and the absorbance (A) was measured at 535 nm and 650 nm. The A535 values were corrected with the A650 values using formula A535 = A535-2.2 × A650. Anthocyanin contents were calculated according to a standard curve prepared with the same protocol.

Quantification of total Pi content

Total Pi content was quantified according to the U.S. Environmental Protection Agency Method 365.2 with minor modifications [39]. Briefly, samples (~50 mg/sample) were flamed to ash after recording of their dry weights and then 100 μL of concentrated HCl was added. Ten microliters of the mixture were drawn and diluted into 790 μL of water, followed by addition of 200 μL assay solution (4.8 mM NH4MoO4, 2.5 N H2SO4, and 35 μM ascorbic acid). The reactions were incubated at 45 °C for 20 min and the absorbance at 650 nm was measured spectrophotometrically. Contents of total Pi were calculated according to the Pi standard curve prepared with the same procedure.

Quantification and staining of acid phosphatase activity

Activity of acid phosphatase was quantified using the pNPP hydrolysis assay according to a previously described method [101]. Briefly, 30 mg of samples were grounded, transferred to Eppendorf tubes and then spin for 10 min at 2000 × g. Reactions containing 100 μL supernatant, 100 μL p-nitrophenol sodium phosphate and 2.8 mL buffer were kept at 30 °C for 10 min, with shaking occasionally, and terminated by addition of 1 mL 0.5 M NaOH. The absorbance of the reactions was determined spectrophotometrically at 400 nm and the activity of acid phosphatase was calculated from the production of p-nitrophenol. Total protein was estimated using Bradford’s reagent and the total acid phosphatase activity was expressed as mU/mg protein. Staining of acid phosphatase activity was performed as described by Tomscha et al. [102] with minor modifications. Ten-day-old seedlings grown in liquid medium were rinsed in –Pi medium, transferred to fresh medium supplemented with Pi (1 mM) or without Pi and then covered with a layer of 0.05 % agarose solution containing 0.008 % 5-bromo-4-chloro-3-indolyl phosphate (BCIP) [103].

Detection and quantification of H2O2

For quantification of H2O2, 30 mg samples from 4-week-old plants were completely ground, followed by addition of 200 μL 20 mM K2HPO4 (pH6.5) phosphate buffer. Quantification of H2O2 was performed using a commericial kit (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufactruer’s recommendation. Detection of superoxide anion was performed by the nitroblue tetrazolium (NBT) staining [104]. Four-week-old seedlings were vacuum infiltrated with 2 ml of 10 mM potassium phosphate buffer (pH 7.5) containing 10 mM NaN3 and 0.1 % NBT for 30 min, cleared by boiling in 96 % ethanol, remained in 50 % ethanol before taking photos.

qRT-PCR analysis of gene expression

Total RNA was extracted using TRIZOL reagent (Invitrogen, Shanghai, China) according to the manufacturer’s instructions. First strand cDNA was synthesized from 500 ng of total RNA using SuperScript III Kit (Invitrogen, Shanghai, China). The qPCR reactions contained 12.5 μL SYBR Premix Ex TaqTM (TaKaRa, Dalian, China), 0.1 μg cDNA and 7.5 pmol of each of gene-specific primers in 25 μL and were conducted on a CFX96 real-time PCR system (BioRad, Hercules, CA, USA). Gene-specific primers used were as followings: BIK1-q-F, 5′-ACT TAT GGG TAC GCC GCG CCT GAG T-3′; BIK1-q-R, 5′-GGC ACG GAC CAC TTG GTC CA-3′; GUS-q-1F, 5′-AGG TGC ACG GGA ATG TTT CG-3′; GUS-q-1R, 5′-TGT GAG AGT CGC AGA ACA TT-3′; PHR1-q-1F, 5′-GTG ATT GGC ATG AAT GGG CTG AC-3′; PHR1-q-1R, 5′-CGC AAT TCC ACA GAC GGA GAA GG-3′; AT4-q-1F, 5′-GAT CGA AGT TGC CCA AAC GA -3′; AT4-q-1R, 5′-GAG CGA TGA AGA TTG CAT GAA G-3′; WRKY75-q-1F, 5′-GAG AAA TCC ACC GAA AAC TTC GAG CAT AT-3′; WRKY75-q-1R, 5′-GCA TGG TTT TTC TTT TCA ACA CAC GTA AAA TGT A-3′; PHT1.2-q-1F, 5′-AGG GCA AGT CCC TCG AAG AAC T-3′; PHT1.2-q-1R, 5′-ATC AAA CAA ACC ACA AAC AAC TCC ACA T-3′; PHT1.4-q-1F, 5′-TTG CTC CTA ATT TTC CTG ATG CT -3′; PHT1.4-q-1R, 5′-TGT GCC GGC CGA AAT CT-3′. Pri-miRNA399a-1F, 5′-TGG CAG GAA ACC ATT ACT TAG ATC T-3′; Pri-miRNA399a-1R, 5′-TCA CTA ATT AAA AGC AAT GCA TAA AGA GA-3′; Pri-miRNA399d-1F, 5′-TTA CTG GGC GAA TAC TCC TAT GG-3′; Pri-miRNA399d-1R, 5′-ATT TTA CTT GCA TAT CTA GCC AAT GC-3′; PHO2-q-1F, 5′-AGG TTT GAA GCT CCA CCC TCA-3′; PHO2-q-1R, 5′-CCC AAG ATG TGA TTG GAG TTC C-3′; UBQ10-q-1F, 5′-GGC CTT GTA TAA TCC CTG ATG AAT AAG-3′; UBQ10-q-1R, 5′-AAA GAG ATA ACA GGA ACG GAA ACA TAG T-3′. Relative gene expression levels were calculated using 2CT method with three independent biological replicates.

Generation of BIK1 pro ::GUS transgenic line and GUS staining

A 2 kb sequence upstream of the BIK1 start codon was PCR amplified using primers AtBIK1-GUS-1F (5′-ATA CTG CAG CTT GTT GAT TGA TTA ATA GAT TAC C-3, a PstI site in bold) and AtBIK1-GUS-1R (5′-GCC GGA TCC AGA ACT GAA GCA AGA ACC CAT C-3′, a BamHI site in bold) and cloned into vector pCAMBIA1301. Transformation of wild-type Col-0 plants was performed using the floral dip infiltration method mediated by Agrobacterium tumefaciens strain GV3101. Plants of T2 generations from kanamycin-resistant transformants were used for GUS histochemical staining [105].

Abbreviations

+Pi, Pi sufficiency; B. cinerea, Botrytis cinerea; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; BIK1, Botrytis-induced kinase1; MKK, mitogen-activated protein kinase kinase; MPK, mitogen-activated protein kinase; NBT, nitroblue tetrazolium; PAMP, pathogen associated molecular pattern; Pht1, PHOSPHATE TRANSPORTER1; Pi, phosphate; −Pi, Pi deficiency; pNPP, p-nitrophenol sodium phosphate; PPC1, phosphoenolpyruvate carboxylase 1; qRT-PCR, quantitative reverse transcription PCR; ROS, reactive oxygen species; WT, wild type

Declarations

Acknowledgement

We are grateful to Dr. Tesfaye Mengiste, Purdue University, USA, for providing the Arabidopsis WT and bik1 seeds used in this study.

Funding

This work was supported by the National Natural Foundation of Sciences (no. 31272028) and the Ph.D. Program Foundation of Ministry of Education of China (no. 20120101110070).

Availability of supporting data

All data supporting the results of this article are included within the article.

Authors’ contributions

HZ and FS designed the study and all experiments; HZ, LH and YH conducted all the molecular biology and physiology experiments; HZ and FS analyzed the data; FS drafted the manuscript with HZ. All authors have read and approved the final version of the manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable. No human or animals were involved in this study.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
College of Life Science, Taizhou University
(2)
National Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University

References

  1. Raghothama KG. Phosphate transport and signaling. Curr Opin Plant Biol. 2000;3:182–7.View ArticlePubMedGoogle Scholar
  2. Lynch JP, Brown KM. Topsoil foraging–an architectural adaptation of plants to low phosphorus availability. Plant Soil. 2001;237:225–37.View ArticleGoogle Scholar
  3. Abel S, Ticconi CA, Delatorre CA. Phosphate sensing in higher plant. Physiol Plant. 2002;115:1–8.View ArticlePubMedGoogle Scholar
  4. Ticconi CA, Abel S. Short on phosphate: plant surveillance and countermeasures. Trends Plant Sci. 2004;9:548–55.View ArticlePubMedGoogle Scholar
  5. Bucher M, Rausch C, Daram P. Molecular and biochemical mechanisms of phosphorus uptake into plants. J Plant Nutr Soil Sci. 2001;164:209–17.View ArticleGoogle Scholar
  6. Raghothama KG, Karthikeyan AS. Phosphate acquisition. Plant Soil. 2005;274:37–49.View ArticleGoogle Scholar
  7. Williamson LC, Ribrioux SPCP, Fitter AH, Leyser HMO. Phosphate availability regulates root system architecture in Arabidopsis. Plant Physiol. 2001;126:875–82.View ArticlePubMedPubMed CentralGoogle Scholar
  8. Ma Z, Bielenberg DG, Brown KM, Lynch JP. Regulation of root hair density by phosphorus availability in Arabidopsis thaliana. Plant Cell Environ. 2001;24:459–67.View ArticleGoogle Scholar
  9. Schmidt W, Schikora A. Different pathways are involved in phosphate and iron stress-induced alterations of root epidermal cell development. Plant Physiol. 2001;125:2078–84.View ArticlePubMedPubMed CentralGoogle Scholar
  10. Lopez-Bucio J, Hern’andez-Abreu E, S’anchez-Calder ’on L, Nieto- Jacobo MF, Simpson J, Herrera-Estrella L. Phosphate availability alters architecture and causes changes in hormone sensitivity in the Arabidopsis root system. Plant Physiol. 2002;129:244–56.View ArticlePubMedPubMed CentralGoogle Scholar
  11. López-Bucio J, Cruz-Ramírez A, Herrera-Estrella A. The role of nutrient availability in regulating root architecture. Curr Opin Plant Biol. 2003;6:280–7.View ArticlePubMedGoogle Scholar
  12. Niu YF, Chai RS, Jin GL, Wang H, Tang CX, Zhang YS. Responses of root architecture development to low phosphorus availability: a review. Ann Bot. 2013;112:391–408.View ArticlePubMedGoogle Scholar
  13. Mimura T. Regulation of phosphate transport and homeostasis in plant cells. Int Rev Cytol. 1999;191:149–200.View ArticleGoogle Scholar
  14. Vance CP, Uhde-Stone C, Allan DL. Phosphorus acquisition and use: critical adaptations by plants for securing a nonrenewable resource. New Phytol. 2003;157:423–47.View ArticleGoogle Scholar
  15. Kobayashi K, Masuda T, Takamiya K, Ohta H. Membrane lipid alteration during phosphate starvation is regulated by phosphate signaling and auxin/cytokinins cross-talk. Plant J. 2006;47:238–48.View ArticlePubMedGoogle Scholar
  16. Oropeza-Aburto A, Cruz-Ramírez A, Acevedo-Hernández GJ, Pérez-Torres CA, Caballero-Pérez J, Herrera-Estrella L. Functional analysis of the Arabidopsis PLDZ2 promoter reveals an evolutionarily conserved low-Pi-responsive transcriptional enhancer element. J Exp Bot. 2012;63:2189–202.View ArticlePubMedGoogle Scholar
  17. Rausch C, Bucher M. Molecular mechanisms of phosphate transport in plants. Planta. 2002;216:23–37.View ArticlePubMedGoogle Scholar
  18. López-Arredondo DL, Leyva-González MA, González-Morales SI, López-Bucio J, Herrera-Estrella L. Phosphate nutrition: improving low-phosphate tolerance in crops. Annu Rev Plant Biol. 2014;65:95–123.View ArticlePubMedGoogle Scholar
  19. Franco-Zorrilla JM, González E, Bustos R, Linhares F, Leyva A, Paz-Ares J. The transcriptional control of plant responses to phosphate limitation. J Exp Bot. 2004;55:285–93.View ArticlePubMedGoogle Scholar
  20. Lin WY, Lin SI, Chiou TJ. Molecular regulators of phosphate homeostasis in plants. J Exp Bot. 2009;60:1427–38.View ArticlePubMedGoogle Scholar
  21. Rouached H, Arpat AB, Poirier Y. Regulation of phosphate starvation responses in plants: signaling players and cross-talks. Mol Plant. 2010;3:288–99.View ArticlePubMedGoogle Scholar
  22. Jain A, Nagarajan VK, Raghothama KG. Transcriptional regulation of phosphate acquisition by higher plants. Cell Mol Life Sci. 2012;69:3207–24.View ArticlePubMedGoogle Scholar
  23. Zhang Z, Liao H, Lucas WJ. Molecular mechanisms underlying phosphate sensing, signaling, and adaptation in plants. J Integr Plant Biol. 2014;56:192–220.View ArticlePubMedGoogle Scholar
  24. Muchhal US, Pardo JM, Raghothama KG. Phosphate transporters from the higher plant Arabidopsis thaliana. Proc Natl Acad Sci U S A. 1996;93:10519–23.View ArticlePubMedPubMed CentralGoogle Scholar
  25. Daram P, Brunner S, Rausch C, Steiner C, Amrhein N, Bucher M. Pht2;1 encodes a low-affinity phosphate transporter from Arabidopsis. Plant Cell. 1999;11:2153–66.View ArticlePubMedPubMed CentralGoogle Scholar
  26. Knappe S, Flügge UI, Fischer K. Analysis of the plastidic phosphate translocator gene family in Arabidopsis and identification of new phosphate translocator-homologous transporters, classified by their putative substrate-binding site. Plant Physiol. 2003;131:1178–90.View ArticlePubMedPubMed CentralGoogle Scholar
  27. Guo B, Irigoyen S, Fowler TB, Versaw WK. Differential expression and phylogenetic analysis suggest specialization of plastid-localized members of the PHT4 phosphate transporter family for photosynthetic and heterotrophic tissues. Plant Signal Behav. 2008;3:784–90.View ArticlePubMedPubMed CentralGoogle Scholar
  28. Shin H, Shin HS, Dewbre GR, Harrison MJ. Phosphate transport in Arabidopsis: Pht1;1 and Pht1;4 play a major role in phosphate acquisition from both low- and high-phosphate environments. Plant J. 2004;39:629–42.View ArticlePubMedGoogle Scholar
  29. Nussaume L, Kanno S, Javot H, Marin E, Pochon N, Ayadi A, Nakanishi TM, Thibaud MC. Phosphate import in plants: Focus on the PHT1 transporters. Front Plant Sci. 2011;2:83.View ArticlePubMedPubMed CentralGoogle Scholar
  30. Remy E, Cabrito TR, Batista RA, Teixeira MC, Sá-Correia I, Duque P. The Pht1;9 and Pht1;8 transporters mediate inorganic phosphate acquisition by the Arabidopsis thaliana root during phosphorus starvation. New Phytol. 2012;195:356–71.View ArticlePubMedGoogle Scholar
  31. Cubero B, Nakagawa Y, Jiang XY, Miura KJ, Li F, Raghothama KG, Bressan RA, Hasegawa PM, Pardo JM. The phosphate transporter PHT4;6 is a determinant of salt tolerance that is localized to the Golgi apparatus of Arabidopsis. Mol Plant. 2009;2:535–52.View ArticlePubMedGoogle Scholar
  32. Hassler S, Lemke L, Jung B, Möhlmann T, Krüger F, Schumacher K, Espen L, Martinoia E, Neuhaus HE. Lack of the Golgi phosphate transporter PHT4;6 causes strong developmental defects, constitutively activated disease resistance mechanisms and altered intracellular phosphate compartmentation in Arabidopsis. Plant J. 2012;72:732–44.View ArticlePubMedGoogle Scholar
  33. Liu J, Yang L, Luan M, Wang Y, Zhang C, Zhang B, et al. A vacuolar phosphate transporter essential for phosphate homeostasis in Arabidopsis. Proc Natl Acad Sci U S A. 2015;112:E6571–8.View ArticlePubMedPubMed CentralGoogle Scholar
  34. Hamburger D, Rezzonico E, MacDonald-Comber Petétot J, Somerville C, Poirier Y. Identification and characterization of the Arabidopsis PHO1 gene involved in phosphate loading to the xylem. Plant Cell. 2002;14:889–902.View ArticlePubMedPubMed CentralGoogle Scholar
  35. Nagarajan VK, Jain A, Poling MD, Lewis AJ, Raghothama KG, Smith AP. Arabidopsis Pht1;5 mobilizes phosphate between source and sink organs and influences the interaction between phosphate homeostasis and ethylene signaling. Plant Physiol. 2011;156:1149–63.View ArticlePubMedPubMed CentralGoogle Scholar
  36. Lapis-Gaza HR, Jost R, Finnegan PM. Arabidopsis PHOSPHATE TRANSPORTER1 genes PHT1;8 and PHT1;9 are involved in root-to-shoot translocation of orthophosphate. BMC Plant Biol. 2014;14:334.View ArticlePubMedPubMed CentralGoogle Scholar
  37. Rubio V, Linhares F, Solano R, Mart’ın AC, Iglesias J, Leyva, Paz-Ares J. A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plants and in unicellular algae. Genes Dev. 2001;15:2122–33.View ArticlePubMedPubMed CentralGoogle Scholar
  38. Chen ZH, Nimmo GA, Jenkins GI, Nimmo HG. BHLH32 modulates several biochemical and morphological processes that respond to Pi starvation in Arabidopsis. Biochem J. 2007;405:191–8.View ArticlePubMedPubMed CentralGoogle Scholar
  39. Devaiah BN, Karthikeyan AS, Raghothama KG. WRKY75 transcription factor is a modulator of phosphate acquisition and root development in Arabidopsis. Plant Physiol. 2007;143:1789–801.View ArticlePubMedPubMed CentralGoogle Scholar
  40. Devaiah BN, Nagarajan VK, Raghothama KG. Phosphate homeostasis and root development in Arabidopsis are synchronized by the zinc finger transcription factor ZAT6. Plant Physiol. 2007;145:147–59.View ArticlePubMedPubMed CentralGoogle Scholar
  41. Devaiah BN, Madhuvanthi R, Karthikeyan AS, Raghothama KG. Phosphate starvation responses and gibberellic acid biosynthesis are regulated by the MYB62 transcription factor in Arabidopsis. Mol Plant. 2009;2:43–58.View ArticlePubMedGoogle Scholar
  42. Chen YF, Li LQ, Xu Q, Kong YH, Wang H, Wu WH. The WRKY6 transcription factor modulates PHOSPHATE1 expression in response to low Pi stress in Arabidopsis. Plant Cell. 2009;21:3554–66.View ArticlePubMedPubMed CentralGoogle Scholar
  43. Wang H, Xu Q, Kong YH, Chen Y, Duan JY, Wu WH, Chen YF. Arabidopsis WRKY45 transcription factor activates PHOSPHATE TRANSPORTER1;1 expression in response to phosphate starvation. Plant Physiol. 2014;164:2020–9.View ArticlePubMedPubMed CentralGoogle Scholar
  44. Ramaiah M, Jain A, Raghothama KG. Ethylene Response Factor070 regulates root development and phosphate starvation-mediated responses. Plant Physiol. 2014;164:1484–98.View ArticlePubMedPubMed CentralGoogle Scholar
  45. Su T, Xu Q, Zhang FC, Chen Y, Li LQ, Wu WH, Chen YF. WRKY42 modulates phosphate homeostasis through regulating phosphate translocation and acquisition in Arabidopsis. Plant Physiol. 2015;167:1579–91.View ArticlePubMedPubMed CentralGoogle Scholar
  46. Chen CY, Wu K, Schmidt W. The histone deacetylase HDA19 controls root cell elongation and modulates a subset of phosphate starvation responses in Arabidopsis. Sci Rep. 2015;5:15708.View ArticlePubMedPubMed CentralGoogle Scholar
  47. Pant BD, Burgos A, Pant P, Cuadros-Inostroza A, Willmitzer L, Scheible WR. The transcription factor PHR1 regulates lipid remodeling and triacylglycerol accumulation in Arabidopsis thaliana during phosphorus starvation. J Exp Bot. 2015;66:1907–18.View ArticlePubMedPubMed CentralGoogle Scholar
  48. Fujii H, Chiou TZ, Lin S-I, Aung K, Zhu J-K. A miRNA involved in phosphate-starvation response in Arabidopsis. Curr Biol. 2005;15:2038–43.View ArticlePubMedGoogle Scholar
  49. Bari R, Pant BD, Stitt M, Scheible WR. PHO2, MicroRNA399, and PHR1 define a phosphate-signaling pathway in plants. Plant Physiol. 2006;141:988–99.View ArticlePubMedPubMed CentralGoogle Scholar
  50. Aung K, Lin SI, Wu CC, Huang YT, Su CL, Chiou TZ. pho2, a phosphate overaccumulator, is caused by a nonsense mutation in a microRNA399 target gene. Plant Physiol. 2006;141:1000–11.View ArticlePubMedPubMed CentralGoogle Scholar
  51. Chiou TJ, Aung K, Lin SI, Wu CC, Chiang SF, Sua CL. Regulation of phosphate homeostasis by microRNA in Arabidopsis. Plant Cell. 2006;18:412–21.View ArticlePubMedPubMed CentralGoogle Scholar
  52. Lin SI, Chiang SF, Lin WY, Chen JW, Tseng CY, Wu PC, Chiou TJ. Regulatory network of microRNA399 and PHO2 by systemic signaling. Plant Physiol. 2008;147:732–46.View ArticlePubMedPubMed CentralGoogle Scholar
  53. Hsieh LC, Lin SI, Shih AC, Chen JW, Lin WY, Tseng CY, Li WH, Chiou TJ. Uncovering small RNA-mediated responses to phosphate deficiency in Arabidopsis by deep sequencing. Plant Physiol. 2010;151:2120–32.View ArticleGoogle Scholar
  54. Miura K, Rus A, Sharkhuu A, Yokoi S, Karthikeyan AS, Raghothama KG, Baek D, Koo YD, Jin JB, Bressan RA, Yun DJ, Hasegawa PM. The Arabidopsis SUMO E3 ligase SIZ1 controls phosphate deficiency responses. Proc Natl Acad Sci U S A. 2005;102:7760–5.View ArticlePubMedPubMed CentralGoogle Scholar
  55. Bayle V, Arrighi JF, Creff A, Nespoulous C, Vialaret J, Rossignol M, Gonzalez E, Paz-Ares J, Nussaume L. Arabidopsis thaliana high-affinity phosphate transporters exhibit multiple levels of posttranslational regulation. Plant Cell. 2011;23:1523–35.View ArticlePubMedPubMed CentralGoogle Scholar
  56. Lei L, Li Y, Wang Q, Xu J, Chen Y, Yang H, et al. Activation of MKK9-MPK3/MPK6 enhances phosphate acquisition in Arabidopsis thaliana. New Phytol. 2014;203:1146–60.View ArticlePubMedGoogle Scholar
  57. Li WF, Perry PJ, Prafulla NN, Schmidt W. Ubiquitin-specific protease 14 (UBP14) is involved in root responses to phosphate deficiency in Arabidopsis. Mol Plant. 2010;3:212–23.View ArticlePubMedGoogle Scholar
  58. Yong-Villalobos L, González-Morales SI, Wrobel K, Gutiérrez-Alanis D, Cervantes-Peréz SA, Hayano-Kanashiro C, Oropeza-Aburto A, Cruz-Ramírez A, Martínez O, Herrera-Estrella L. Methylome analysis reveals an important role for epigenetic changes in the regulation of the Arabidopsis response to phosphate starvation. Proc Natl Acad Sci U S A. 2015;112:E7293–302.View ArticlePubMedPubMed CentralGoogle Scholar
  59. López-Bucio J, Hernández-Abreu E, Sánchez-Calderón L, Pérez-Torres A, Rampey RA, Bartel B, Herrera-Estrella L. An auxin transport independent pathway is involved in phosphate stress-induced root architectural alterations in Arabidopsis. Identification of BIG as a mediator of auxin in pericycle cell activation. Plant Physiol. 2005;137:681–91.View ArticlePubMedPubMed CentralGoogle Scholar
  60. Nacry P, Canivenc G, Muller B, Azmi A, Van Onckelen H, Rossignol M, Doumas P. A role for auxin redistribution in the responses of the root system architecture to phosphate starvation in Arabidopsis. Plant Physiol. 2005;138:2061–74.View ArticlePubMedPubMed CentralGoogle Scholar
  61. Grieneisen VA, Xu J, Mar’ee AFM, Hogeweg P, Scheres B. Auxin transport is sufficient to generate a maximum and gradient guiding root growth. Nature. 2007;449:1008–13.View ArticlePubMedGoogle Scholar
  62. Martin AC, del Pozo JC, Iglesias J, Rubio V, Solano R, de la Pena A, Paz-Ares J. Influence of cytokinins on the expression of phosphate starvation responsive genes in Arabidopsis. Plant J. 2000;24:559–67.View ArticlePubMedGoogle Scholar
  63. Wang X, Yi K, Tao Y, Wang F, Wu Z, Jiang D, Chen X, Zhou L, Wu P. Cytokinin represses phosphate-starvation response through increasing of intracellular phosphate level. Plant Cell Environ. 2006;29:1924–35.View ArticlePubMedGoogle Scholar
  64. Ma Z, Baskin TI, Brown KM, Lynch JP. Regulation of root elongation under phosphorus stress involves changes in ethylene responsiveness. Plant Physiol. 2003;131:1381–90.View ArticlePubMedPubMed CentralGoogle Scholar
  65. Zhang Y, Lynch JP, Brown K. Ethylene and phosphorus availability have interacting yet distinct effects on root hair development. J Exp Bot. 2003;54:2351–61.View ArticlePubMedGoogle Scholar
  66. Nagarajan VK, Smith AP. Ethylene’s role in phosphate starvation signaling: more than just a root growth regulator. Plant Cell Physiol. 2012;53:277–86.View ArticlePubMedGoogle Scholar
  67. Jiang CF, Gao XH, Liao LL, Harberd NP, Fu XD. Phosphate starvation root architecture and anthocyanin accumulation responses are modulated by the gibberellin-DELLA signaling pathway in Arabidopsis. Plant Physiol. 2007;145:1460–70.View ArticlePubMedPubMed CentralGoogle Scholar
  68. Veronese P, Nakagami H, Bluhm B, Abuqamar S, Chen X, Salmeron J, Dietrich RA, Hirt H, Mengiste T. The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens. Plant Cell. 2006;18:257–73.View ArticlePubMedPubMed CentralGoogle Scholar
  69. Lu D, Wu S, Gao X, Zhang Y, Shan L, He P. A receptor-like cytoplasmic kinase, BIK1, associates with a flagellin receptor complex to initiate plant innate immunity. Proc Natl Acad Sci U S A. 2010;107:496–501.View ArticlePubMedGoogle Scholar
  70. Zhang J, Li W, Xiang T, Liu Z, Laluk K, Ding X, Dietrich RA, Hirt H, Mengiste T. Receptor-like cytoplasmic kinases integrate signaling from multiple plant immune receptors and are targeted by a Pseudomonas syringae effector. Cell Host Microbe. 2010;7:290–301.Google Scholar
  71. Laluk K, Luo H, Chai M, Dhawan R, Lai Z, Mengiste T. Biochemical and genetic requirements for function of the immune response regulator BOTRYTIS-INDUCED KINASE1 in plant growth, ethylene signaling, and PAMP-triggered immunity in Arabidopsis. Plant Cell. 2011;23:2831–49.View ArticlePubMedPubMed CentralGoogle Scholar
  72. Liu Z, Wu Y, Yang F, Zhang Y, Chen S, Xie Q, Tian X, Zhou JM. BIK1 interacts with PEPRs to mediate ethylene-induced immunity. Proc Natl Acad Sci U S A. 2013;110:6205–10.View ArticlePubMedPubMed CentralGoogle Scholar
  73. Lin W, Lu D, Gao X, Jiang S, Ma X, Wang Z, Mengiste T, He P, Shan L. Inverse modulation of plant immune and brassinosteroid signaling pathways by the receptor-like cytoplasmic kinase BIK1. Proc Natl Acad Sci U S A. 2013;110:12114–9.View ArticlePubMedPubMed CentralGoogle Scholar
  74. Lei J, A Finlayson S, Salzman RA, Shan L, Zhu-Salzman K. BOTRYTIS-INDUCED KINASE1 modulates Arabidopsis resistance to green peach aphids via PHYTOALEXIN DEFICIENT4. Plant Physiol. 2014;165:1657–70.View ArticlePubMedPubMed CentralGoogle Scholar
  75. Péret B, Clément M, Nussaume L, Desnos T. Root developmental adaptation to phosphate starvation: better safe than sorry. Trends Plant Sci. 2011;16:442–50.View ArticlePubMedGoogle Scholar
  76. Mittler R, Vanderauwera S, Gollery M, Van Breusegem F. Reactive oxygen gene network of plants. Trends Plant Sci. 2004;9:490–8.View ArticlePubMedGoogle Scholar
  77. Foyer CH, Noctor G. Redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and physiological responses. Plant Cell. 2005;17:1866–75.View ArticlePubMedPubMed CentralGoogle Scholar
  78. Shin H, Shin HS, Chen R, Harrison MJ. Loss of At4 function impacts phosphate distribution between the roots and the shoots during phosphate starvation. Plant J. 2006;45:712–26.View ArticlePubMedGoogle Scholar
  79. Misson J, Raghothama KG, Jain A, Jouhet J, Block MA, Bligny R, Ortet P, Creff A, Somerville S, Rolland N, et al. A genome-wide transcriptional analysis using Arabidopsis thaliana Affymetrix gene chips determined plant responses to phosphate deprivation. Proc Natl Acad Sci U S A. 2005;102:11934–9.View ArticlePubMedPubMed CentralGoogle Scholar
  80. Lan P, Li W, Schmidt W. Genome-wide co-expression analysis predicts protein kinases as important regulators of phosphate deficiency-induced root hair remodeling in Arabidopsis. BMC Genomics. 2013;14:210.View ArticlePubMedPubMed CentralGoogle Scholar
  81. Gregory A, Hurley BA, Tran HT, Valentine AJ, She YM, Knowles VL, Plaxton WC. In vivo regulatory phosphorylation of the phosphoenolpyruvate carboxylase AtPPC1 in phosphate-starved Arabidopsis thaliana. Biochem J. 2009;420:57–65.Google Scholar
  82. Sanchez-Calderon L, Lopez-Bucio J, Chacon-Lopez A, Cruz-Ramırez A, Nieto-Jacobo F, Dubrovsky JG, Herrera-Estrella L. Phosphate starvation induces a determinate development program in root of Arabidopsis thaliana. Plant Cell Physiol. 2005;46:174–84.View ArticlePubMedGoogle Scholar
  83. Plaxton WC, Tran HT. Metabolic adaptations of phosphate starved plants. Plant Physiol. 2011;156:1006–15.View ArticlePubMedPubMed CentralGoogle Scholar
  84. Hurley BA, Tran HT, Marty NJ, Park J, Snedden WA, Mullen RT, Mullen RT, Plaxton WC The dual-targeted purple acid phosphatase isozyme AtPAP26 is essential for efficient acclimation of Arabidopsis to nutritional phosphate deprivation. Plant Physiol. 2010;153:1112–22.Google Scholar
  85. Robinson WD, Park J, Tran HT, Del Vecchio HA, Ying S, Zins JL, Patel K, McKnight TD, Plaxton WC. The secreted purple acid phosphatase isozymes AtPAP12 and AtPAP26 play a pivotal role in extracellular phosphate-scavenging by Arabidopsis thaliana. J Exp Bot. 2012;63:6531–42.View ArticlePubMedPubMed CentralGoogle Scholar
  86. Del Vecchio HA, Ying S, Park J, Knowles VL, Kanno S, Tanoi K, et al. The cell wall-targeted purple acid phosphatase AtPAP25 is critical for acclimation of Arabidopsis thaliana to nutritional phosphorus deprivation. Plant J. 2014;80:569–81.View ArticlePubMedGoogle Scholar
  87. Tran HT, Plaxton WC. Proteomic analysis of alterations in the secretome of Arabidopsis thaliana suspension cells subjected to nutritional phosphate deficiency. Proteomics. 2008;8:4317–26.View ArticlePubMedGoogle Scholar
  88. Tran HT, Qian W, Hurley BA, She YM, Wang D, Plaxton WC. Biochemical and molecular characterization of AtPAP12 and AtPAP26: the predominant purple acid phosphatase isozymes secreted by phosphate-starved Arabidopsis thaliana. Plant Cell Environ. 2010;33:1789–803.View ArticlePubMedGoogle Scholar
  89. Torres MA. ROS in biotic interactions. Physiol Plant. 2010;138:414–29.View ArticlePubMedGoogle Scholar
  90. Suzuki N, Koussevitzky S, Mittler R, Miller G. ROS and redox signalling in the response of plants to abiotic stress. Plant Cell Environ. 2012;35:259–70.View ArticlePubMedGoogle Scholar
  91. Baxter A, Mittler R, Suzuki N. ROS as key players in plant stress signalling. J Exp Bot. 2013;65:1229–40.View ArticlePubMedGoogle Scholar
  92. Shin R, Berg RH, Schachtman DP. Reactive oxygen species and root hairs in Arabidopsis root response to nitrogen, phosphorus and potassium deficiency. Plant Cell Physiol. 2005;46:1350–7.View ArticlePubMedGoogle Scholar
  93. Foreman J, Demidchik V, Bothwell JH, Mylona P, Miedema H, Torres MA, Linstead P, Costa S, Brownlee C, Jones JD, DaviesJM, Dolan L. Reactive oxygen species produced by NADPH oxidase regulate plant cell growth. Nature. 2003;422:442–6.Google Scholar
  94. Rentel MC, Lecourieux D, Ouaked F, Usher SL, Petersen L, Okamoto H, Knight H, Peck SC, Grierson CS, Hirt H, Knight MR. OXI1 kinase is necessary for oxidative burst-mediated signalling in Arabidopsis. Nature. 2004;427:858–61.View ArticlePubMedGoogle Scholar
  95. Tyburski J, Dunajska K, Tretyn A. Reactive oxygen species localization in roots of Arabidopsis thaliana seedlings under phosphate deficiency. Plant Growth Reg. 2009;59:27–36.View ArticleGoogle Scholar
  96. Chacón-López A, Ibarra-Laclette E, Sánchez-Calderón L, Gutiérrez-Alanis D, Herrera-Estrella L. Global expression pattern comparison between low phosphorus insensitive 4 and WT Arabidopsis reveals an important role of reactive oxygen species and jasmonic acid in the root tip response to phosphate starvation. Plant Signal Behav. 2011;6:382–92.View ArticlePubMedPubMed CentralGoogle Scholar
  97. Kadota Y, Sklenar J, Derbyshire P, Stransfeld L, Asai S, Ntoukakis V, Jones JD, Shirasu K, Menke F, Jones A, Zipfel C. Direct regulation of the NADPH oxidase RBOHD by the PRR-associated kinase BIK1 during plant immunity. Mol Cell. 2014;54:43–55.View ArticlePubMedGoogle Scholar
  98. Li L, Li M, Yu L, Zhou Z, Liang X, Liu Z, Jones JD, Shirasu K, Menke F, Jones A, Zipfel C. The FLS2-associated kinase BIK1 directly phosphorylates the NADPH oxidase RbohD to control plant immunity. Cell Host Microbe. 2014;15:329–38.Google Scholar
  99. Karthikeyan AS, Varadarajan DK, Mukatira UT, D’Urzo MP, Damz B, Raghothama KG. Regulated expression of Arabidopsis phosphate transporters. Plant Physiol. 2002;130:221–33.View ArticlePubMedPubMed CentralGoogle Scholar
  100. Lange H, Shropshire W, Mohr H. An analysis of phytochrome-mediated anthocyanin synthesis. Plant Physiol. 1971;47:649–55.View ArticlePubMedPubMed CentralGoogle Scholar
  101. Richardson AE, Hadobas PA, Hayes JE. Extracellular secretion of Aspergillus phytase from Arabidopsis roots enables plants to obtain phosphorus from phytate. Plant J. 2001;25:641–9.View ArticlePubMedGoogle Scholar
  102. Tomscha JL, Trull MC, Deikman J, Lynch JP, Guiltinan MJ. Phosphatase under-producer mutants have altered phosphorus relations. Plant Physiol. 2004;135:334–45.View ArticlePubMedPubMed CentralGoogle Scholar
  103. Trull MC, Deikman J. An Arabidopsis mutant missing one acid phosphatase isoform. Planta. 1998;206:544–50.View ArticlePubMedGoogle Scholar
  104. Doke N. Involvement of superoxide anion generation in the hypersensitive response of potato tuber tissue to infection with an incompatible race of Phytophthora infestans and to the hyphal wall components. Physiol Plant Pathol. 1983;23:345–57.View ArticleGoogle Scholar
  105. Jefferson RA, Kavanagh TA, Bevan MW. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 1987;6:3901–7.PubMedPubMed CentralGoogle Scholar

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